SUICIDE INHIBITION OF ONCOGENIC K-RAS G12C PROCEEDS VIA SHIFT TO THE INACTIVE CONFORMATION

Significance of the topic

The Ras family of small GTPases controls key signaling pathways in cell growth and differentiation. Mutations in K-Ras, notably G12C, lock the protein in active states and drive oncogenesis in many cancers. Despite decades of interest, direct Ras inhibitors have been elusive. Understanding how covalent inhibitors alter K-Ras G12C structure is critical for designing drugs that effectively suppress malignant signaling.

Objectives and study overview

This study applies hydrogen–deuterium exchange mass spectrometry (HX-MS) to compare three states of K-Ras G12C: bound to GDP (inactive), to the nonhydrolyzable GTP analog GMPPNP (active), and to the covalent GDP–mimetic SML-8-73-1. The main aim is to determine whether the inhibitor shifts the protein toward the inactive conformation or induces unique structural features.

Methodology and instrumentation

Deuterium labeling was performed at room temperature by exposing the three protein forms to D2O over time intervals from seconds to hours. After quenching, samples underwent online pepsin digestion and UPLC separation. A Waters Q-Tof Premier mass spectrometer with ESI source recorded mass spectra. Peptide identification used tandem MS in Waters PLGS and MSE modes, and deuterium uptake was analyzed with DynamX software.

Main results and discussion

• Coverage: Over 40 peptides provided 94% sequence coverage.
• Exchange kinetics: Most regions exhibited EX2 behavior (gradual exchange), while the switch I region (residues 7–20) displayed EX1 kinetics (cooperative exchange) under active and inhibited conditions.
• Switch I protection: Deuterium uptake in residues 7–20 was significantly lower in the GDP and SML-8-73-1 states than with GMPPNP, indicating stabilization of an inactive-like conformation by the inhibitor.
• Other regions: Residues adjacent to the nucleotide and guanosine moiety (114–120) showed minor uptake differences, while the remainder of the protein was similar across all states.
• Conformational ensemble: Isotopic distributions suggest heterogeneous populations when K-Ras is active, but a single, more rigid ensemble when bound to GDP or inhibitor.

Benefits and practical applications

• Mechanistic insight: Confirms suicide inhibitor SML-8-73-1 stabilizes the inactive GDP-bound fold of K-Ras G12C.
• Drug design: HX-MS can guide optimization of covalent inhibitors by pinpointing dynamic hotspots and confirming target engagement.
• Analytical strategy: Demonstrates the power of HX-MS to dissect conformational effects of small-molecule drugs on challenging targets.

Future trends and opportunities

• Structure–dynamics profiling: Combining HX-MS with computational modeling and cryo-EM to map transient states and allosteric networks.
• Broader inhibitor classes: Applying similar workflows to noncovalent and reversible Ras inhibitors, as well as other GTPases.
• High-throughput HX-MS: Automation and microfluidic devices could allow screening of compound libraries for conformational stabilization snapshots.

Conclusion

The HX-MS analysis reveals that covalent binding of SML-8-73-1 drives K-Ras G12C toward an inactive conformation comparable to GDP-bound protein, particularly by constraining the switch I region. This study underscores the relevance of conformational dynamics in drug action and offers a robust analytical framework for next-generation Ras inhibitors.

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